A pipeline for rapidly evaluating activity and inferring mechanisms of action of prospective antifungal compounds.

BACKGROUND: Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS: The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS: Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Volatile organic compounds of the soil bacterium Bacillus halotolerans suppress pathogens and elicit defense-responsive genes in plants.

Volatile organic compounds (VOCs) produced by bacteria play an important, yet relatively unexplored role in interactions between plants and phytopathogens. In this study, the soil bacterium Bacillus halotolerans NYG5 was identified as a potent biocontrol agent against several phytopathogenic fungi (Macrophomina phaseolina, Rhizoctonia solani, Pythium aphanidermatum, and Sclerotinia sclerotiorum) through the production of VOCs. NYG5-emitted VOCs also inhibited the growth of bacterial pathogens (Agrobacterium tumefaciens, Xanthomonas campestris, Clavibacter michiganensis, and Pseudomonas syringae). When cultured in various growth media, NYG5 produced a variety of VOCs. Five distinct VOCs (2-methylbutanoic acid, 5-methyl-2-hexanone, 2,3-hexanedione, 2-ethyl-1-hexanol, and 6-methyl-2-heptanone) were identified using headspace GC-MS. 2,3-Hexanedione exhibited potent lethal effects on the tested phytopathogens and nematicidal activity against Meloidogyne javanica at a concentration of 50 ppm. In addition, 0.05 ppm 2,3-hexanedione stimulated the expression of pathogenesis-related genes 1 and 2 in Arabidopsis thaliana. Interestingly, 2,3-hexanedione is used as a food additive at higher concentrations than those tested in this study. Hence, 2,3-hexanedione is a promising biologically active compound that might serve as a sustainable alternative to common chemical pesticides and an elicitor of plant defense.

Corrigendum: Comparative genomics of Bacillus cereus sensu lato spp. biocontrol strains in correlation to in-vitro phenotypes and plant pathogen antagonistic capacity.

[This corrects the article DOI: 10.3389/fmicb.2023.996287.].

Comparative genomics of Bacillus cereus sensu lato spp. biocontrol strains in correlation to in-vitro phenotypes and plant pathogen antagonistic capacity.

Bacillus cereus sensu lato (Bcsl) strains are widely explored due to their capacity to antagonize a broad range of plant pathogens. These include B. cereus sp. UW85, whose antagonistic capacity is attributed to the secondary metabolite Zwittermicin A (ZwA). We recently isolated four soil and root-associated Bcsl strains (MO2, S-10, S-25, LSTW-24) that displayed different growth profiles and in-vitro antagonistic effects against three soilborne plant pathogens models: Pythium aphanidermatum (oomycete) Rhizoctonia solani (basidiomycete), and Fusarium oxysporum (ascomycete). To identify genetic mechanisms potentially responsible for the differences in growth and antagonistic phenotypes of these Bcsl strains, we sequenced and compared their genomes, and that of strain UW85 using a hybrid sequencing pipeline. Despite similarities, specific Bcsl strains had unique secondary metabolite and chitinase-encoding genes that could potentially explain observed differences in in-vitro chitinolytic potential and anti-fungal activity. Strains UW85, S-10 and S-25 contained a (~500 Kbp) mega-plasmid that harbored the ZwA biosynthetic gene cluster. The UW85 mega-plasmid contained more ABC transporters than the other two strains, whereas the S-25 mega-plasmid carried a unique cluster containing cellulose and chitin degrading genes. Collectively, comparative genomics revealed several mechanisms that can potentially explain differences in in-vitro antagonism of Bcsl strains toward fungal plant pathogens.

Bacterial volatile organic compounds as biopesticides, growth promoters and plant-defense elicitors: Current understanding and future scope.

Bacteria emit a large number of volatile organic compounds (VOCs) into the environment. VOCs are species-specific and their emission depends on environmental conditions, such as growth medium, pH, temperature, incubation time and interaction with other microorganisms. These VOCs can enhance plant growth, suppress pathogens and act as signaling molecules during plant-microorganism interactions. Some bacterial VOCs have been reported to show strong antimicrobial, nematicidal, pesticidal, plant defense, induced tolerance and plant-growth-promoting activities under controlled conditions. Commonly produced antifungal VOCs include dimethyl trisulfide, dimethyl disulfide, benzothiazole, nonane, decanone and 1-butanol. Species of Bacillus, Pseudomonas, Arthrobacter, Enterobacter and Burkholderia produce plant growth promoting VOCs, such as acetoin and 2,3-butenediol. These VOCs affect expression of genes involved in defense and development in plant species (i.e., Arabidopsis, tobacco, tomato, potato, millet and maize). VOCs are also implicated in altering pathogenesis-related genes, inducing systemic resistance, modulating plant metabolic pathways and acquiring nutrients. However, detailed mechanisms of action of VOCs need to be further explored. This review summarizes the bioactive VOCs produced by diverse bacterial species as an alternative to agrochemicals, their mechanism of action and challenges for employment of bacterial VOCs for sustainable agricultural practices. Future studies on technological improvements for bacterial VOCs application under greenhouse and open field conditions are warranted.

Short- and long-term effects of continuous compost amendment on soil microbiome community.

Organic amendment, and especially the use of composts, is a well-accepted sustainable agricultural practice. Compost increases soil carbon and microbial biomass, changes enzymatic activity, and enriches soil carbon and nitrogen stocks. However, relatively little is known about the immediate and long-term temporal dynamics of agricultural soil microbial communities following repeated compost applications. Our study was conducted at two field sites: Newe Ya'ar (NY, Mediterranean climate) and Gilat (G, semi-arid climate), both managed organically over 4 years under either conventional fertilization (0, zero compost) or three levels of compost amendment (20, 40 and 60 m3/ha or 2, 4, 6 L/m2). Microbial community dynamics in the soils was examined by high- and low-time-resolution analyses. Annual community composition in compost-amended soils was significantly affected by compost amendment levels in G (first, second and third years) and in NY (third year). Repeated sampling at high resolution (9-10 times over 1 year) showed that at both sites, compost application initially induced a strong shift in microbial communities, lasting for up to 1 month, followed by a milder response. Compost application significantly elevated alpha diversity at both sites, but differed in the compost-dose correlation effect. We demonstrate higher abundance of taxa putatively involved in organic decomposition and characterized compost-related indicator taxa and a compost-derived core microbiome at both sites. Overall, this study describes temporal changes in the ecology of soil microbiomes in response to compost vs. conventional fertilization.

Freshwater microbial metagenomes sampled across different water body characteristics, space and time in Israel.

Freshwater bodies are critical components of terrestrial ecosystems. The microbial communities of freshwater ecosystems are intimately linked water quality. These microbes interact with, utilize and recycle inorganic elements and organic matter. Here, we present three metagenomic sequence datasets (total of 182.9 Gbp) from different freshwater environments in Israel. The first dataset is from diverse freshwater bodies intended for different usages - a nature reserve, irrigation and aquaculture facilities, a tertiary wastewater treatment plant and a desert rainfall reservoir. The second represents a two-year time-series, collected during 2013-2014 at roughly monthly intervals, from a water reservoir connected to an aquaculture facility. The third is from several time-points during the winter and spring of 2015 in Lake Kinneret, including a bloom of the cyanobacterium Microcystis sp. These datasets are accompanied by physical, chemical, and biological measurements at each sampling point. We expect that these metagenomes will facilitate a wide range of comparative studies that seek to illuminate new aspects of freshwater microbial ecosystems and inform future water quality management approaches.

Spike Formation Is a Turning Point Determining Wheat Root Microbiome Abundance, Structures and Functions.

Root selection of their associated microbiome composition and activities is determined by the plant's developmental stage and distance from the root. Total gene abundance, structure and functions of root-associated and rhizospheric microbiomes were studied throughout wheat growth season under field conditions. On the root surface, abundance of the well-known wheat colonizers Proteobacteria and Actinobacteria decreased and increased, respectively, during spike formation, whereas abundance of Bacteroidetes was independent of spike formation. Metagenomic analysis combined with functional co-occurrence networks revealed a significant impact of plant developmental stage on its microbiome during the transition from vegetative growth to spike formation. For example, gene functions related to biofilm and sensorial movement, antibiotic production and resistance and carbons and amino acids and their transporters. Genes associated with these functions were also in higher abundance in root vs. the rhizosphere microbiome. We propose that abundance of transporter-encoding genes related to carbon and amino acid, may mirror the availability and utilization of root exudates. Genes related to antibiotic resistance mechanisms were abundant during vegetative growth, while after spike formation, genes related to the biosynthesis of various antibiotics were enriched. This observation suggests that during root colonization and biofilm formation, bacteria cope with competitor's antibiotics, whereas in the mature biofilm stage, they invest in inhibiting new colonizers. Additionally, there is higher abundance of genes related to denitrification in rhizosphere compared to root-associated microbiome during wheat growth, possibly due to competition with the plant over nitrogen in the root vicinity. We demonstrated functional and phylogenetic division in wheat root zone microbiome in both time and space: pre- and post-spike formation, and root-associated vs. rhizospheric niches. These findings shed light on the dynamics of plant-microbe and microbe-microbe interactions in the developing root zone.

Pairwise Interactions of Three Related Pseudomonas Species in Plant Roots and Inert Surfaces.

Bacteria are social organisms that interact extensively within and between species while responding to external stimuli from their environments. Designing synthetic microbial communities can enable efficient and beneficial microbiome implementation in many areas. However, in order to design an efficient community, one must consider the interactions between their members. Using a reductionist approach, we examined pairwise interactions of three related Pseudomonas species in various microenvironments including plant roots and inert surfaces. Our results show that the step between monoculture and co-culture is already very complex. Monoculture root colonization patterns demonstrate that each isolate occupied a particular location on wheat roots, such as root tip, distance from the tip, or scattered along the root. However, pairwise colonization outcomes on the root did not follow the bacterial behavior in monoculture, suggesting various interaction patterns. In addition, we show that interspecies interactions on a microscale on inert surface take part in co-culture colonization and that the interactions are affected by the presence of root extracts and depend on its source. The understanding of interrelationships on the root may contribute to future attempts to manipulate and improve bacterial colonization and to intervene with root microbiomes to construct and design effective synthetic microbial consortia.

Elevated CO2 and nitrate levels increase wheat root-associated bacterial abundance and impact rhizosphere microbial community composition and function.

Elevated CO2 stimulates plant growth and affects quantity and composition of root exudates, followed by response of its microbiome. Three scenarios representing nitrate fertilization regimes: limited (30 ppm), moderate (70 ppm) and excess nitrate (100 ppm) were compared under ambient and elevated CO2 (eCO2, 850 ppm) to elucidate their combined effects on root-surface-associated bacterial community abundance, structure and function. Wheat root-surface-associated microbiome structure and function, as well as soil and plant properties, were highly influenced by interactions between CO2 and nitrate levels. Relative abundance of total bacteria per plant increased at eCO2 under excess nitrate. Elevated CO2 significantly influenced the abundance of genes encoding enzymes, transporters and secretion systems. Proteobacteria, the largest taxonomic group in wheat roots (~ 75%), is the most influenced group by eCO2 under all nitrate levels. Rhizobiales, Burkholderiales and Pseudomonadales are responsible for most of these functional changes. A correlation was observed among the five gene-groups whose abundance was significantly changed (secretion systems, particularly type VI secretion system, biofilm formation, pyruvate, fructose and mannose metabolism). These changes in bacterial abundance and gene functions may be the result of alteration in root exudation at eCO2, leading to changes in bacteria colonization patterns and influencing their fitness and proliferation.

Temporal Resistome and Microbial Community Dynamics in an Intensive Aquaculture Facility with Prophylactic Antimicrobial Treatment.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

Correction to: The microbiome as a biosensor: functional profiles elucidate hidden stress in hosts.

An amendment to this paper has been published and can be accessed via the original article.

The microbiome as a biosensor: functional profiles elucidate hidden stress in hosts.

BACKGROUND: Microbial communities are highly responsive to environmental cues, and both their structure and activity can be altered in response to changing conditions. We hypothesized that host-associated microbial communities, particularly those colonizing host surfaces, can serve as in situ sensors to reveal environmental conditions experienced by both microorganisms and the host. For a proof-of-concept, we studied a model plant-soil system and employed a non-deterministic gene-centric approach. A holistic analysis was performed using plants of two species and irrigation with water of low quality to induce host stress. Our analyses examined the genetic potential (DNA) and gene expression patterns (RNA) of plant-associated microbial communities, as well as transcriptional profiling of host plants. RESULTS: Transcriptional analysis of plants irrigated with treated wastewater revealed significant enrichment of general stress-associated root transcripts relative to plants irrigated with fresh water. Metagenomic analysis of root-associated microbial communities in treated wastewater-irrigated plants, however, revealed enrichment of more specific stress-associated genes relating to high levels of salt, high pH and lower levels of oxygen. Meta-analysis of these differentially abundant genes obtained from other metagenome studies, provided evidence of the link between environmental factors such as pH and oxygen and these genes. Analysis of microbial transcriptional response demonstrated that enriched gene content was actively expressed, which implies contemporary response to elevated levels of pH and salt. CONCLUSIONS: We demonstrate here that microbial profiling can elucidate stress signals that cannot be observed even through interrogation of host transcriptome, leading to an alternate mechanism for evaluating in situ conditions experienced by host organisms. This study is a proof-of-concept for the use of microbial communities as microsensors, with great potential for interrogation of a wide range of host systems. Video Abstract.

Effects of tillage practices on soil microbiome and agricultural parameters.

No-tillage (NT) is a common soil-conservation management practice with known agricultural advantages and drawbacks. However, its short- and long-term effects on the soil microbiome have not been well established. Here, we compared conventional (CT), minimal (MT) and NT practices in two agricultural fields in the north of Israel over a period of 3 years. Edaphic properties, plant-associated pests, weed species abundance and soil microbial community structure were assessed to examine the effects of tillage. Tillage significantly altered physical and chemical soil properties, and a significant increase in hydrolytic and redox microbial activities was observed in NT soils from both sites. Consistent with this, the microbial community structure of NT samples diverged significantly over time from those of CT samples. Repetitive tillage and even a single tillage event caused significant changes in the relative abundance of microorganisms at taxonomic levels ranging from phylum to OTU. However, no significant difference between treatments was found in microbial community alpha-diversity or crop yield. Conversely, higher levels of weed diversity and some pests number were found in NT samples. Overall, we demonstrate that tillage plays a major role in shaping microbial community structure, and in influencing additional environmental, ecological and agricultural soil parameters.

Altering N2O emissions by manipulating wheat root bacterial community.

Nitrous oxide (N2O) is a greenhouse gas and a potent ozone-depleting substance in the stratosphere. Agricultural soils are one of the main global sources of N2O emissions, particularly from cereal fields due to their high areal coverage. The aim of this study was to isolate N2O-reducing bacteria able to mitigate N2O emissions from the soil after inoculation. We isolated several bacteria from wheat roots that were capable of N2O reduction in vitro and studied their genetic potential and activity under different environmental conditions. Three of these isolates- all carrying the nitrous oxide reductase-encoding clade I nosZ, able to reduce N2O in vitro, and efficient colonizers of wheat roots- presented different N2O-reduction strategies when growing in the root zone, possibly due to the different conditions in situ and their metabolic preferences. Each isolate seemed to prefer to operate at different altered oxygen levels. Isolate AU243 (related to Agrobacterium/Rhizobium) could reduce both nitrate and N2O and operated better at lower oxygen levels. Isolate AU14 (related to Alcaligenes faecalis), lacking nitrate reductases, operated better under less anoxic conditions. Isolate NT128 (related to Pseudomonas stutzeri) caused slightly increased N2O emissions under both anoxic and ambient conditions. These results therefore emphasize the importance of a deep understanding of soil-plant-microbe interactions when environmental application is being considered.

Root microbiome response to treated wastewater irrigation.

With increasing fresh water (FW) scarcity, the use of treated wastewater (TWW) for crop irrigation is expanding globally. Besides clear benefits, some undesired long-term effects of irrigation with this low quality water on plant performance have been reported. As the rhizosphere microbiome can mediate plant-soil interactions, an examination of the response of these organisms to TWW is necessary to understand the full effects of water quality. In the current study, the effects of irrigation water quality on the microbial community structure of soil and roots as well as edaphic properties and plant performance were evaluated. We compared soil and roots microbiomes of two different plant species (tomato and lettuce), each grown in two distinct soils, and irrigated with either FW or TWW. Irrigation with TWW significantly increase soil pH, EC, K, Na and DOC, and decrease plant fruit and shoot weight, relatively to samples irrigated with FW. We calculated the effect size of plant species, soil type, and irrigation water quality on microbial community structure in soil and root. In the roots, plant species and irrigation water were the dominant factors in shaping both total (DNA based) and active (RNA based) microbial communities, with both factors contributing similarly to the observed microbial population. Soil type and irrigation water were the dominant factors shaping the total microbial community in the soil and were of similar magnitude. Irrigation water quality is demonstrated to be a major force in shaping root-associated microbiome, leading to altered microbial community structure in the critical juncture between plant and soil.

Quality of Irrigation Water Affects Soil Functionality and Bacterial Community Stability in Response to Heat Disturbance.

Anthropogenic activities alter the structure and function of a bacterial community. Furthermore, bacterial communities structured by the conditions the anthropogenic activities present may consequently reduce their stability in response to an unpredicted acute disturbance. The present mesocosm-scale study exposed soil bacterial communities to different irrigation water types, including freshwater, fertilized freshwater, treated wastewater, and artificial wastewater, and evaluated their response to a disturbance caused by heat. These effectors may be considered deterministic and stochastic forces common in agricultural operations of arid and semiarid regions. Bacterial communities under conditions of high mineral and organic carbon availability (artificial wastewater) differed from the native bacterial community and showed a proteobacterial dominance. These bacterial communities had a lower resistance to the heat treatment disturbance than soils under conditions of low resource availability (high-quality treated wastewater or freshwater). The latter soil bacterial communities showed a higher abundance of operational taxonomic units (OTUs) classified as Bacilli These results were elucidated by soil under conditions of high resource availability, which lost higher degrees of functional potential and had a greater bacterial community composition change. However, the functional resilience, after the disturbance ended, was higher under a condition of high resource availability despite the bacterial community composition shift and the decrease in species richness. The functional resilience was directly connected to the high growth rates of certain Bacteroidetes and proteobacterial groups. A high stability was found in samples that supported the coexistence of both resistant OTUs and fast-growing OTUs.IMPORTANCE This report presents the results of a study employing a hypothesis-based experimental approach to reveal the forces involved in determining the stability of a soil bacterial community to disturbance. The resultant postdisturbance bacterial community composition dynamics and functionality were analyzed. The paper demonstrates the relatedness of community structure and stability under cultivation conditions prevalent in an arid area under irrigation with water of different qualities. The use of common agricultural practices to demonstrate these features has not been described before. The combination of a fundamental theoretical issue in ecology with common and concerning disturbances caused by agricultural practice makes this study unique. Furthermore, the results of the present study have applicable importance regarding soil conservation, as it enables a better characterization and monitoring of stressed soil bacterial communities and possible intervention to reduce the stress. It will also be of valued interest in coming years, as fresh water scarcity and the use of alternative water sources are expected to rise globally.

Host Specificity and Spatial Distribution Preference of Three Pseudomonas Isolates.

Plant hosts recruit and maintain a distinct root-associated microbiota based on host and bacterium traits. However, past studies disregarded microbial strain-host specificity and spatial micro-heterogeneity of the root compartment. Using genetic manipulation, confocal laser scanning microscopy, real-time quantitative PCR, and genome sequencing we characterized the colonization patterns of three Pseudomonas spp. isolates native to wheat roots, on the micro-scale. Namely, isolates P. fluorescens NT0133, P. stutzeri NT124, and P. stutzeri NT128. All three isolates preferentially colonized wheat over cucumber roots that served as control for host specificity. Furthermore, not only had the isolates strong host specificity but each isolate had a distinct spatial distribution on the root, all within a few millimeters. Isolate P. stutzeri-NT0124 preferentially colonized root tips, whereas P. fluorescens-NT0133 showed a preference for zones distant from the tip. In contrast, isolate P. stutzeri-NT0128 had no preference for a specific niche on the root. While all isolates maintained genetic potential for motility and biofilm formation their phenotype varied significantly and corresponded to their niche preference. These results demonstrate the importance of spatial colonization patterns, governed by both niche and bacterial characteristics which will have great importance in future attempts to manipulate the plant microbiome by constructing synthetic microbial consortia.

Analysis of Microbial Functions in the Rhizosphere Using a Metabolic-Network Based Framework for Metagenomics Interpretation.

Advances in metagenomics enable high resolution description of complex bacterial communities in their natural environments. Consequently, conceptual approaches for community level functional analysis are in high need. Here, we introduce a framework for a metagenomics-based analysis of community functions. Environment-specific gene catalogs, derived from metagenomes, are processed into metabolic-network representation. By applying established ecological conventions, network-edges (metabolic functions) are assigned with taxonomic annotations according to the dominance level of specific groups. Once a function-taxonomy link is established, prediction of the impact of dominant taxa on the overall community performances is assessed by simulating removal or addition of edges (taxa associated functions). This approach is demonstrated on metagenomic data describing the microbial communities from the root environment of two crop plants - wheat and cucumber. Predictions for environment-dependent effects revealed differences between treatments (root vs. soil), corresponding to documented observations. Metabolism of specific plant exudates (e.g., organic acids, flavonoids) was linked with distinct taxonomic groups in simulated root, but not soil, environments. These dependencies point to the impact of these metabolite families as determinants of community structure. Simulations of the activity of pairwise combinations of taxonomic groups (order level) predicted the possible production of complementary metabolites. Complementation profiles allow formulating a possible metabolic role for observed co-occurrence patterns. For example, production of tryptophan-associated metabolites through complementary interactions is unique to the tryptophan-deficient cucumber root environment. Our approach enables formulation of testable predictions for species contribution to community activity and exploration of the functional outcome of structural shifts in complex bacterial communities. Understanding community-level metabolism is an essential step toward the manipulation and optimization of microbial function. Here, we introduce an analysis framework addressing three key challenges of such data: producing quantified links between taxonomy and function; contextualizing discrete functions into communal networks; and simulating environmental impact on community performances. New technologies will soon provide a high-coverage description of biotic and a-biotic aspects of complex microbial communities such as these found in gut and soil. This framework was designed to allow the integration of high-throughput metabolomic and metagenomic data toward tackling the intricate associations between community structure, community function, and metabolic inputs.

Evidence of Increased Antibiotic Resistance in Phylogenetically-Diverse Aeromonas Isolates from Semi-Intensive Fish Ponds Treated with Antibiotics.

The genus Aeromonas is ubiquitous in aquatic environments encompassing a broad range of fish and human pathogens. Aeromonas strains are known for their enhanced capacity to acquire and exchange antibiotic resistance genes and therefore, are frequently targeted as indicator bacteria for monitoring antimicrobial resistance in aquatic environments. This study evaluated temporal trends in Aeromonas diversity and antibiotic resistance in two adjacent semi-intensive aquaculture facilities to ascertain the effects of antibiotic treatment on antimicrobial resistance. In the first facility, sulfadiazine-trimethoprim was added prophylactically to fingerling stocks and water column-associated Aeromonas were monitored periodically over an 11-month fish fattening cycle to assess temporal dynamics in taxonomy and antibiotic resistance. In the second facility, Aeromonas were isolated from fish skin ulcers sampled over a 3-year period and from pond water samples to assess associations between pathogenic strains to those in the water column. A total of 1200 Aeromonas isolates were initially screened for sulfadiazine resistance and further screened against five additional antimicrobials. In both facilities, strong correlations were observed between sulfadiazine resistance and trimethoprim and tetracycline resistances, whereas correlations between sulfadiazine resistance and ceftriaxone, gentamicin, and chloramphenicol resistances were low. Multidrug resistant strains as well as sul1, tetA, and intI1 gene-harboring strains were significantly higher in profiles sampled during the fish cycle than those isolated prior to stocking and these genes were extremely abundant in the pathogenic strains. Five phylogenetically distinct Aeromonas clusters were identified using partial rpoD gene sequence analysis. Interestingly, prior to fingerling stocking the diversity of water column strains was high, and representatives from all five clusters were identified, including an A. salmonicida cluster that harbored all characterized fish skin ulcer samples. Subsequent to stocking, diversity was much lower and most water column isolates in both facilities segregated into an A. veronii-associated cluster. This study demonstrated a strong correlation between aquaculture, Aeromonas diversity and antibiotic resistance. It provides strong evidence for linkage between prophylactic and systemic use of antibiotics in aquaculture and the propagation of antibiotic resistance.